Biology subjects

Zimmer, G.

Publications and source records attributed to Zimmer, G..

15 recordsLinked to original sources

bioRxiv2026

Genetic modification of primary airway epithelial cell cultures defines MX1 as a barrier for zoonotic influenza A viruses in the human respiratory epithelium

The antiviral innate immune response is a major species barrier that emerging viruses must overcome to infect and transmit efficiently in new hosts. In humans, the interferon-stimulated MX1 gene encodes the antiviral MX1 protein, which restricts zoonotic influenza A viruses (IAVs). Resistance to MX1 is considered a key feature of human-adapted IAVs and a potential determinant of zoonotic and pandemic risk. However, MX1-mediated restriction and viral escape have mostly been investigated in cell lines and animal models, which incompletely recapitulate virus-host interactions in the human airway epithelium, representing the primary site of IAV replication. Although primary human airway epithelial cell (hAEC) cultures, such as airway organoids and air-liquid interface (ALI) cultures, provide a physiologically relevant model, their use in studying MX1 restriction has been limited by challenges in achieving efficient and reliable genetic modification. Here, we used engineered virus-like particles (eVLPs) to deliver Cas9-sgRNA ribonucleoprotein complexes into primary hAECs and disrupt MX1 expression. This approach generated MX1-knockout airway organoids and organoid-derived hAEC ALI cultures with >90% genomic-level editing efficiency and near-complete loss of MX1 protein, while preserving epithelial differentiation at the air-liquid interface. MX1 knockout in hAEC ALI cultures had only a minor effect on the replication kinetics of the human-adapted H1N1pdm09 strain. By contrast, MX1 knockout markedly impaired interferon lambda-mediated restriction of the zoonotic bovine H5N1/Texas and avian H7N9/Anhui strains, resulting in viral titers up to 3-4 logs TCID50 higher than in MX1-competent cultures. These findings provide direct evidence that MX1 restricts zoonotic IAVs in primary human airway epithelium. Our study establishes a biologically relevant platform for assessing the sensitivity of emerging IAVs to a key antiviral host factor and thereby informs evaluations into their zoonotic and pandemic risk.

microbiology↗

bioRxiv2026

Bovine-derived influenza A virus (H5N1) shows efficient replication in well-differentiated human nasal epithelial cells without requiring genetic adaptation

Highly pathogenic avian influenza (H5N1) viruses of clade 2.3.4.4b have caused significant losses among bird populations worldwide and have repeatedly crossed the species barrier, infecting mammals, including humans. However, efficient human-to-human transmission has not yet been observed. Here, we demonstrate that an H5N1 virus isolated from bovine milk in Texas in 2024 (H5N1Tex/24) replicates in differentiated human nasal epithelial cells as efficiently as a 2009 pandemic H1N1 virus strain (H1N1HH4/09) at both 37 {degrees}C and 33 {degrees}C. The adaptive mutations PB2-M631L and PA-K497R do not affect H5N1Tex/24 replication at 37 {degrees}C but promote replication at 33 {degrees}C. Conversely, H5N1BE/22, a virus from the same clade isolated from a pelican in 2022 that lacks these mutations, replicates in human nasal epithelial cells at 37 {degrees}C as efficiently as H5N1Tex/24 but exhibits limited replication at 33 {degrees}C. Introducing the two mutations PB2-M631L and PA-K497R did not overcome this limitation. Furthermore, nasal epithelial cells express receptors for both human and avian influenza viruses. Accordingly, no mutations were detected in HA which are known to switch receptor preference. Finally, we demonstrate that H5N1Tex/24 remains sensitive to the antiviral effects of interferon-{lambda} (IFN-{lambda}), however, infected nasal epithelial cells secrete only small amounts of this cytokine. Overall, our results suggest that H5N1Tex/24 possesses intrinsic traits enabling efficient replication in the human upper airways.

microbiology↗

bioRxiv2025

Non-cytolytic re-engineering of a viral vaccine vector enables durable effector-memory T cell immunity by reinforcing type I IFN induction

Replication-deficient viral vector systems hold promise for CD8 T cell-based vaccination, but the molecular mechanisms accounting for platform-specific differences in immunogenicity remain ill- defined. When comparing prototypic single-cycle vaccine delivery platforms we found that lymphocytic choriomeningitis virus-based vectors (rLCMV), which are non-cytolytic, elicited more durable and effector-memory-differentiated CD8 T cell responses than vectors based on cytolytic vesicular stomatitis virus (rVSV). Hence we re-engineered rVSV to be non-cytolytic (rVSVMq). This vector induced more durable and effector-differentiated CD8 T cell memory than the parental rVSV and it afforded superior protection against Listeria challenge. Improved CD8 T cell responses of non-cytolytic rVSVMq were driven by a reinforced type I interferon (IFN-I) response and its direct sensing by vaccination-induced CD8 T cells. Many vector cargo-specific CD8 T cells in the splenic marginal zone of rVSVMq- or rLCMV-vaccinated mice were in contact with vector cargo-expressing cells that co- expressed type I interferon. In contrast, rVSV-vectored cargo-expressing contacts of specific CD8 T cells were largely IFN type I-negative. Thereby, vaccination with non-cytolytic viral vectors offered an opportunity for CD8 T cells to integrate peptide-MHC and IFN-I signals during priming. These mechanistic insights should help to refine vaccines aimed at eliciting durable and protective effector- memory CD8 T cell immunity.

immunology↗

bioRxiv2025

An RNA replicon vaccine encoding HA and NA prevents shedding of antigen-drifted 2009 pandemic H1N1 influenza virus in the pig model

Seasonal influenza viruses escape the human immune response by antigenic drift, i.e. the positive selection of point mutations that prevent the binding of inhibitory antibodies to the influenza antigens HA and NA. The efficacy of seasonal influenza vaccines can be less than 50% if the selected influenza vaccine strain does not match the antigenic characteristics of the circulating seasonal influenza virus. In this study, we used the porcine model to evaluate the efficacy of an RNA replicon vaccine encoding the HA and NA antigens of A/Hamburg/4/2009 (H1N1) (H1N1HH4/09) in inducing cross-reactive immunity. We found that a single intramuscular immunization with this vaccine elicited high levels of antibodies with H1N1HH4/09-neutralizing activity and potent N1-sialidase inhibition. A second immunization with the same H1/N1 RNA replicon particles or with a live-attenuated influenza vaccine (LAIV) based on a modified H1N1HH4/09 virus boosted the inhibitory activity of the immune sera against the antigen-drifted A/Victoria/2570/2019 (H1N1) (H1N1Vic/19) strain. Interestingly, vaccination elicited N1-specific antibodies that also inhibited the activity of avian N1 sialidase and potently inhibited the replication of A/cattle/Texas/063224-24-1/2024 (H5N1) (H5N1Tex/24) in vitro. When challenged nasally with a H1N1HH4/09 /H1N1Vic/19 6:2 reassortant virus encoding the HA and NA antigens of H1N1Vic/19, immunized pigs did not shed infectious virus while the control animals did, suggesting that homologous prime/boost vaccination with H1/N1 replicon particles can block virus replication in the upper respiratory tract as efficiently as the heterologous RNA replicon prime/LAIV boost immunization regimen. In conclusion, RNA replicons encoding both HA and NA either used alone or in combination with LAIV mediate protection against antigen-drifted influenza viruses and reduce the risk of vaccination breakthroughs due to antigen mismatch. Furthermore, this vaccine may also limit the infection by zoonotic H5N1 viruses.

microbiology↗

bioRxiv2025

Impact of pH and temperature in dairy processing on the infectivity of H5N1 avian influenza viruses

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 (clade 2.3.4.4b) have crossed the species barrier and caused a mastitis-like infection in dairy cows. The high levels of infectious virus found in the milk raised considerable concerns about the safety of raw milk products. This study examined the effect of temperature and pH on the stability of HPAIV and low-pathogenic avian influenza viruses (LPAIV). We found that H5N1 HPAIV remained infectious in milk at 4{degrees}C for four weeks, with slow decreases at 21{degrees}C, and complete inactivation at 37{degrees}C after four weeks. H5N1 LPAIV was stable at 50{degrees}C for 30 minutes but inactivated at higher temperatures (55{degrees}C for 10 minutes, 60{degrees}C for 1 minute, or 72{degrees}C for 30 seconds). At pH levels between 6 and 10, the virus remained stable but was partially inactivated at pH 5.0 and completely inactivated at pH 4.0. During yogurt production, H5N1 LPAIV was completely inactivated when the pH reached 4.3. In cheese production, the lowest pH reached was between 5.0 and 5.3. When H5N1 LPAIV was incubated with soft and semi-hard cheese for one day at 4 {degrees}C, infectious virus titers decreased by 5.1 and 3.9 log10, respectively. When H5N1 LPAIV was incubated with buffer adjusted to pH 5.0, infectious virus titer dropped by only 3.3 log10, suggesting that, alongside pH, other processes of cheese ripening likely influence virus stability. In conclusion, H5N1 avian influenza viruses are largely inactivated during lactic acid fermentation of raw milk. Future studies will assess the required cheese ripening time for complete inactivation.

microbiology↗

bioRxiv2025

Immunization with a novel RNA replicon vaccine confers long-lasting protection against H5N1 avian influenza virus in 24 bird species

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 (clade 2.3.4.4b) have spread worldwide and caused the death of hundreds of millions of wild birds and domestic poultry. Moreover, spill over of H5N1 HPAIV from infected birds to more than 50 different mammalian species including humans has been recorded. While, licensed vaccines for protection of avian or mammalian species are not yet available, a few candidate vaccines are being trialled. Here, we report on the experimental vaccination of chickens and captive wild birds using a propagation-defective vesicular stomatitis virus (VSV), in which the essential envelope glycoprotein (G) protein gene was replaced by a modified hemagglutinin gene derived from a clade 2.3.4.4b H5N1 isolated in 2022 in the animal park of Bern, Switzerland. VSV{Delta}G(H5mb) was produced on helper cells providing the VSV G protein in trans. Specific pathogen-free (SPF) chickens that were immunized twice via the intramuscular route with adjuvant-free VSV{Delta}G(H5mb) replicon particles induced high levels of virus-neutralizing serum antibodies and were fully protected against lethal infection by H5N1 HPAIV (clade 2.3.4.4b). Notably, immunized animals did not shed challenge virus from the respiratory or gastrointestinal tract, suggesting that herd immunity can be achieved. The same vaccine was used to immunize a total of 317 captive wild birds at Bern Animal Park and Zoo Basel, representing 24 different species. No vaccine-associated side effects were observed. Birds without previous contact to H5Nx viruses produced high to very high H5-specific neutralizing antibody titers following the second immunization, while birds showing H5-specific antibodies prior to vaccination, already developed high neutralising antibody titers after a single immunization. One year after vaccination, most animals still showed significant neutralizing antibody titers, indicating that VSV{Delta}G(H5mb) is able to induce a long-lasting protective immune response. Our results indicate that VSV{Delta}G(H5mb) is an extraordinary safe and highly efficacious vaccine to stop H5N1 replication in various avian species.

microbiology↗

bioRxiv2024

Aging shapes infection profiles of influenza A virus and SARS-CoV-2 in human lung slices

The recent coronavirus disease 2019 (COVID-19) outbreak revealed the susceptibility of elderly patients to respiratory virus infections, showing cell senescence or subclinical persistent inflammatory profiles and favouring the development of severe pneumonia. In our study, we evaluated the potential influence of lung aging on the efficiency of replication of influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as determined the pro-inflammatory and antiviral responses of the distal lung tissue. Using precision-cut lung slices (PCLS) from donors of different ages, we found that pandemic H1N1 and avian H5N1 IAV replicated in the lung parenchyma with high efficacy. In contrast to these IAV strains, SARS-CoV-2 early isolate and Delta variant of concern (VOC) replicated less efficiently in PCLS. Interestingly, both viruses showed reduced replication in PCLS from older compared to younger donors, suggesting that aged lung tissue represents a sub-optimal environment for viral replication. Regardless of the age-dependent viral loads, PCLS responded to infection with both viruses by an induction of IL-6 and IP-10/CXCL10 mRNAs, being highest for H5N1. Finally, while SARS-CoV-2 infection was not causing detectable cell death, IAV infection caused significant cytotoxicity and induced significant early interferon responses. In summary, our findings suggest that aged lung tissue might not favour viral dissemination, pointing to a determinant role of dysregulated immune mechanisms in the development of severe disease. New & NoteworthyPCLS from donors of varying ages were exposed to SARS-CoV-2 or IAV. Notably, the latter exhibited the highest replication efficacy, triggering early interferon responses, elevated IL-6 and IP-10/CXCL10 mRNAs expression, and significant cell death compared to SARS-CoV-2. Overall, across all age groups, the pulmonary environment showed sustained immunocompetence. For both viruses, older donor-derived PCLS displayed reduced viral permissiveness, suggesting aged lung tissue might not favour viral dissemination, implying other factors contribute to severe disease development.

microbiology↗

bioRxiv2024

Intramuscular prime/intranasal boost vaccination to induce sterilizing immunity against influenza A virus infection

The most commonly used influenza vaccines are made from inactivated viruses and are administered via the intramuscular route. Although these vaccines can protect from severe lower respiratory tract disease, they do not completely prevent virus replication in the upper respiratory tract, and this may lead to virus excretion and dissemination. Therefore, nasally administered live-attenuated influenza vaccines (LAIV) that induce mucosal immunity have been developed, but finding an optimal balance between sufficient attenuation and immunogenicity remained challenging. These problems apply to both human and swine influenza vaccines. We have recently developed an LAIV candidate based on the 2009 pandemic H1N1 virus which encodes a truncated NS1 protein and lacks PA-X protein expression (NS1(1-126)-{Delta}PAX). This virus showed a blunted replication and elicited a strong innate immune response. In the present study, we took advantage of the pig animal model to evaluate this vaccine candidate in vivo and to identify a strategy for its improvement. Nasal infection of pigs with the NS1(1-126)-{Delta}PAX LAIV candidate did not cause disease but was associated with prolonged virus shedding from the upper respiratory tract. To increase safety of the vaccine candidate, we developed a novel prime/boost vaccination strategy consisting of a haemagglutinin-encoding propagation-defective vesicular stomatitis virus replicon vaccine for primary immunization via the intramuscular route, and the NS1(1-126)-{Delta}PAX LAIV for secondary immunization via the nasal route. This immunization strategy significantly reduced LAIV shedding, increased the production of specific serum IgG, neutralizing antibodies, Th1 memory cells, and induced virus-specific mucosal IgG and IgA. Of particular note, the immune response induced by this vaccination strategy completely blocked replication of the homologous challenge virus in the respiratory tract, indicating that sterilizing immunity was achieved. In summary, our novel intramuscular prime/intranasal boost vaccine combines the features of high efficacy and safety which are urgently needed to combat influenza epidemics and pandemics. Author summaryInactivated influenza vaccines which are administered intramuscularly are safe but offer only limited protection. In addition, they do not adequately prevent virus transmission by infected individuals. On the other hand, nasally administered live-attenuated influenza vaccines induce a mucosal immune response, which can effectively prevent primary infection and virus excretion. However, live-attenuated vaccines might not be sufficiently immunogenic if they are too attenuated or they trigger a robust immune response but are still too virulent. To overcome this challenge, we have developed a novel prime/boost vaccination strategy consisting of an initial intramuscular immunization with a propagation-defective RNA virus vector and a subsequent nasal immunization with a modified influenza virus that has lost its ability to counteract the hosts innate immune response. Using the pig model, we demonstrate that this approach elicited a more robust immune response both systemically and at mucosal surfaces. Importantly, replication of the vaccine virus in the respiratory tract was reduced, and challenge virus remained undetectable. In summary, our innovative vaccine, which combines intramuscular and intranasal routes of application, demonstrates high efficacy and safety and represents a valuable tool to control influenza epidemics and pandemics.

immunology↗

bioRxiv2022

Non-neutralizing antibody responses to vesicular stomatitis virus-vectored influenza A virus vaccines correlate with protection

Seasonal influenza virus infections remain a major global public health burden. In addition, influenza A virus (IAV) exhibits high pandemic potential through zoonotic spread from avian hosts to humans. Currently licensed IAV vaccines are mainly directed against the immuno-dominant surface protein hemagglutinin (HA). Since HA is antigenically highly variable, IAV can escape vaccine-derived immunity through antigenic drift. However, vaccine preparations such as the live-attenuated influenza vaccine (LAIV) also contain other more conserved viral antigens, whose contribution to influenza immunity are not fully elucidated. To determine the extent to which conserved LAIV antigens contribute to establishing protective immunity against heterologous IAV strains, we generated vesicular stomatitis virus-based single-round vector vaccines expressing individual LAIV antigens, and tested their ability to protect mice from a heterologous challenge with two IAV strains, PR8[H1N1] and rSC35M[H7N7]. We found that immunization with nucleoprotein (NP), ion channel M2, and the stem-region of HA (HAstem), but not matrix protein (M1), provide protection from severe disease caused by either IAV strain. This effect correlated with development of non-neutralizing antibodies cross-reactive with both virus strains. Notably, the individual antigens induced specific IgG subclass profiles with different reactivity against PR8 and rSC35M. Sera from vaccinated animals activated Fc-gamma receptor IV-mediated effector functions, suggesting that they can induce cell-mediated immune defense mechanisms, such as antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Combination of the most potent antigens NP and M2 in a mixed vaccination resulted in enhanced protection against IAV challenge, suggesting that the antibody responses against these antigens were synergistic. Our results demonstrate the potency of NP and M2 proteins to serve as conserved antigen targets, resulting in broad protection against severe IAV disease.

immunology↗

bioRxiv2022

Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior protective humoral immune response to SARS-CoV-2

Immunization with vesicular stomatitis virus (VSV)-vectored COVID-19 vaccine candidates expressing the SARS-CoV-2 spike protein in place of the VSV glycoprotein relies implicitly on expression of the ACE2 receptor at the muscular injection site. Here, we report that such a viral vector vaccine did not induce protective immunity following intramuscular immunization of K18-hACE2 transgenic mice. However, when the viral vector was trans-complemented with the VSV glycoprotein, intramuscular immunization resulted in high titers of spike-specific neutralizing antibodies. The vaccinated animals were fully protected following infection with a lethal dose of SARS-CoV-2-SD614G via the nasal route, and partially protected if challenged with the SARS-CoV-2Delta variant. While dissemination of the challenge virus to the brain was completely inhibited, replication in the lung with consequent lung pathology was not entirely controlled. Thus, intramuscular immunization was clearly enhanced by trans-complementation of the VSV-vectored vaccines by the VSV glycoprotein and led to protection from COVID-19, although not achieving sterilizing immunity.

immunology↗

bioRxiv2021

A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

Over the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) - SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 - has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

microbiology↗

bioRxiv2020

No evidence for human monocyte-derived macrophage infection and antibody-mediated enhancement of SARS-CoV-2 infection

Vaccines are essential to control the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and to protect the vulnerable population. However, one safety concern of vaccination is the possible development of antibody-dependent enhancement (ADE) of SARS-CoV-2 infection. The potential infection of Fc receptor bearing cells such as macrophages, would support continued virus replication and inflammatory responses, and thereby potentially worsen the clinical outcome of COVID-19. Here we demonstrate that SARS-CoV-2 and SARS-CoV-1 neither infect human monocyte-derived macrophages nor induce inflammatory cytokines in these cells, in sharp contrast to Middle East respiratory syndrome (MERS) coronavirus and the common cold human coronavirus 229E. Furthermore, serum from convalescent COVID-19 patients neither induced enhancement of SARS-CoV-2 infection nor innate immune response in human macrophages. These results support the view that ADE may not be involved in the immunopathological processes associated with COVID-19, however, more studies are necessary to understand the potential contribution of antibodies-virus complexes with other cells expressing FcR receptors.

immunology↗

bioRxiv2020

Highly potent bispecific sybodies neutralize SARS-CoV-2

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair (Sb#15 and Sb#68) that can bind simultaneously to the SARS-CoV-2 spike-RBD and efficiently neutralize pseudotyped and live-viruses by interfering with ACE2 interaction. Two spatially-discrete epitopes identified by cryo-EM translated into the rational design of bispecific and tri-bispecific fusions constructs, exhibiting up to 100- and 1000-fold increase in neutralization potency. Cryo-EM of the sybody-spike complex further revealed a novel up-out RBD conformation. While resistant viruses emerged rapidly in the presence of single binders, no escape variants were observed in presence of the bispecific sybody. The multivalent bispecific constructs further increased the neutralization potency against globally-circulating SARS- CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the development of clinically relevant therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

biochemistry↗

bioRxiv2020

LY6E impairs coronavirus fusion and confers immune control of viral disease

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized2. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV.

microbiology↗

bioRxiv2019

T-cell immunoglobulin and mucin (TIM) contributes to Hantaan virus entry into human airway epithelial cells

Hantaviruses are rodent-borne haemorrhagic fever viruses leading to serious diseases. Viral attachment and entry represent the first steps in virus transmission and are promising targets for antiviral therapeutic intervention. Here we investigated receptor use in human airway epithelium of the Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV). Using a biocontained recombinant vesicular stomatitis virus pseudotype platform, we provide first evidence for a role of the cellular phosphatidylserine (PS) receptors of the T-cell immunoglobulin and mucin (TIM) in HTNV and ANDV entry. In line with previous studies, HTNV, but not ANDV, was able to use the glycosaminoglycan heparan sulfate and v{beta}3 integrin as co-receptors. In sum, our studies demonstrate for the first time that hantaviruses use PS receptors and hence apoptotic mimicry to invade human airway epithelium, which may explain why these viruses can easily break the species barrier.

microbiology↗